Thermodynamic and kinetic basis for recognition and repair of 8-oxoguanine in DNA by human 8-oxoguanine-DNA glycosylase

We have used a stepwise increase in ligand complexity approach to estimate the relative contributions of the nucleotide units of DNA containing 7,8-dihydro-8-oxoguanine (oxoG) to its total affinity for human 8-oxoguanine DNA glycosylase (OGG1) and construct thermodynamic models of the enzyme interaction with cognate and non-cognate DNA. Non-specific OGG1 interactions with 10–13 nt pairs within its DNA-binding cleft provides approximately 5 orders of magnitude of its affinity for DNA (ΔG° approximately −6.7 kcal/mol). The relative contribution of the oxoG unit of DNA (ΔG° approximately −3.3 kcal/mol) together with other specific interactions (ΔG° approximately −0.7 kcal/mol) provide approximately 3 orders of magnitude of the affinity. Formation of the Michaelis complex of OGG1 with the cognate DNA cannot account for the major part of the enzyme specificity, which lies in the kcat term instead; the rate increases by 6–7 orders of magnitude for cognate DNA as compared with non-cognate one. The kcat values for substrates of different sequences correlate with the DNA twist, while the KM values correlate with ΔG° of the DNA fragments surrounding the lesion (position from −6 to +6). The functions for predicting the KM and kcat values for different sequences containing oxoG were found

DNA polymerase I (Klenow fragment): Role of the structure and length of a template in enzyme recognition

AbstractThe values of Kd and Gibbs energy (ΔG°) have been measured for complexes of the template site of DNA polymerase I Klenow fragment with the homo-oligonucleotides d(pC)n, d(pT)n, d(pG)n and d(pA)n and hetero-oligonucleotides of various structures and lengths. These parameters were evaluated from the protective effect of the oligonucleotide on enzyme inactivation by the affinity reagents d(Tp)2C[Pt2+(NH3)2OH](pT)7 and d[(Tp)2C(Pt2+(NH3)2OH)p]3T of the template site. The present results and previously reported data [(1985) Biorg. Khim. 13, 357–369] indicate that the nucleoside components of the template form complexes as a result of their hydrophobic interactions with the enzyme. Only one template internucleotide phosphate forms an Me2+-dependent electrostatic contact and a hydrogen bond with the enzyme. The 19–20-nucleotide fragments of the template appear to interact with the protein molecule

Sequence dependence of isothermal DNA amplification via EXPAR

Isothermal nucleic acid amplification is becoming increasingly important for molecular diagnostics. Therefore, new computational tools are needed to facilitate assay design. In the isothermal EXPonential Amplification Reaction (EXPAR), template sequences with similar thermodynamic characteristics perform very differently. To understand what causes this variability, we characterized the performance of 384 template sequences, and used this data to develop two computational methods to predict EXPAR template performance based on sequence: a position weight matrix approach with support vector machine classifier, and RELIEF attribute evaluation with Naïve Bayes classification. The methods identified well and poorly performing EXPAR templates with 67–70% sensitivity and 77–80% specificity. We combined these methods into a computational tool that can accelerate new assay design by ruling out likely poor performers. Furthermore, our data suggest that variability in template performance is linked to specific sequence motifs. Cytidine, a pyrimidine base, is over-represented in certain positions of well-performing templates. Guanosine and adenosine, both purine bases, are over-represented in similar regions of poorly performing templates, frequently as GA or AG dimers. Since polymerases have a higher affinity for purine oligonucleotides, polymerase binding to GA-rich regions of a single-stranded DNA template may promote non-specific amplification in EXPAR and other nucleic acid amplification reactions

Influence of forage crop rotations on crop yields and phytosanitary conditions of soils in the Baikal region

The article considers the practical aspects of ensuring the preservation of natural ecosystems based on the use of organic farming technologies. It presents the results of research in the forest-steppe zone of the Baikal region for 2011-2018. On the basis of the phytosanitary state of crops studying analysis, it was found that the correct alternation of crops in crop rotation suppresses the level of weeds, thereby not affecting the productivity of agricultural crops. The paper reveals the characteristics of agrophysical and water properties of gray forest soil, the influence of forage crop rotations with meadow clover on the yield of cultivated crops. According to the research results, the authors found that overseeding of legumes in the fields of forage crop rotations increases the average productivity by 16.6% in comparison with the control option. The influence of the aftereffect of perennial legumes in crop rotations increases the yield of grain fodder and silage crops by 20-31.8%. The crop rotation with two fields of meadow clover (crop rotation No. 3) was determined to be the best for all indicators